A NOVEL PATTERN OF EMBRYONIC NUTRITION IN A VIVIPAROUS REPTILE

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1 J. exp. Biol. 174, (1993) Printed in Great Britain The Company of Biologists Limited A NOVEL PATTERN OF EMBRYONIC NUTRITION IN A VIVIPAROUS REPTILE BY JAMES R. STEWART AND MICHAEL B. THOMPSON Faculty of Biological Science, The University of Tulsa, Tulsa, Oklahoma 74104, USA and School of Biological Sciences, The University of Sydney, New South Wales 2006, Australia Accepted 15 September 1992 Summary Vitellogenesis and placental transfer both contribute substantially to embryonic nutrition in the viviparous scincid lizard Pseudemoiaentrecasteauxii. Neonatal wet mass was 396% greater than egg wet mass and neonatal dry mass was 168% greater than egg dry mass. We estimate that 49% of the organic molecules for embryonic growth and metabolism were provided by placental nourishment. This pattern of embryonic nutrition, in which ovarian and uterine contributions are approximately equivalent, has not been reported previously for reptiles. Female Bassiana duperreyi, a closely related oviparous species, produced larger clutches of larger eggs. Although there was a great disparity in egg mass between these two species, differences in newborn composition were less pronounced. Mean dry mass, adjusted for female size, of B. duperreyi eggs was 135% greater than mean dry mass of P. entrecasteauxii eggs, yet mean dry mass of B. duperreyi offspring was only 21% greater than that of P. entrecasteauxii offspring. Embryonic gains in water and inorganic salts during gestation in P. entrecasteauxii were substantial and resulted in the production of neonates that contained greater quantities of these variables compared to offspring of B. duperreyi. These data confirm Weekes hypothesis that P. entrecasteauxii is matrotrophic and support her hypothesis that this species has undergone evolutionary reduction in yolk quantity. Introduction The evolution of viviparity among vertebrates has involved two phenomena: shifts in the way in which young are produced and in the method of provisioning developing young with nourishment. The reproductive patterns of vertebrate taxa display a variety of combinations of these two distinct reproductive processes (Blackburn et al. 1985; Wourms et al. 1988; Wake, 1989; Blackburn, 1992; Wourms and Lombardi, 1992). With respect to the first of these, vertebrates either lay eggs (oviparity) or give birth to young (viviparity) (Wake, 1989; Blackburn, 1992; Wourms and Lombardi, 1992). Developing embryos may be nourished from yolk deposited in the ovary during vitellogenesis Key words: viviparity, placentotrophy, lecithotrophy, placentation, fetal nutrition, Pseudemoia entrecasteauxii, Bassiana duperreyi.

2 98 J. R. STEWART AND M. B. THOMPSON (lecithotrophy) or may be nourished by alternative pathways (matrotrophy) (Wourms, 1981; Blackburn et al. 1985). Intrauterine gestation with parturition of independent neonates (viviparity) is a prominent reproductive mode among squamate reptiles (Blackburn, 1982, 1985; Shine, 1985). Sources of embryonic nutrition among these species include both yolk (lecithotrophy) and a form of matrotrophy, placental transfer (placentotrophy). Two primary patterns of nutrient provision are known (Blackburn, 1992; Stewart, 1992). Many species ovulate yolk-rich eggs that provide the primary nourishment to developing embryos, while the uterus also acts as an important source of nutrients. Functionally, these species are primarily lecithotrophic, although embryonic nutrition is supplemented by placentotrophy. In contrast, other species have a large neonate to egg mass ratio, indicating a second pattern of embryonic nourishment characterized by predominant or perhaps complete dependence on placental nutrition (Blackburn et al. 1984; Vitt and Blackburn, 1983; Ghiara et al. 1987; Blackburn and Vitt, 1992). These two recognized patterns are distinct yet might be described as ends of a continuum. Sources of embryonic nutrition for oviparous squamates are less well known and only two lizard species have been studied (Packard et al. 1985; Florian, 1990). No information about patterns of embryonic nutrition is available for closely related species with different reproductive modes and thus hypotheses for the evolution of patterns of fetal nutrition have not been based on historical relationships. Australian scincid lizards historically placed within the genus Leiolopisma (Mittleman, 1952; Greer, 1974) have long figured prominently in hypotheses concerning the evolution of viviparity and placentation among Squamata (Weekes, 1935). The significance of this group is based on the presence of both oviparous and viviparous species as well as the existence of variation in placental structure (Weekes, 1935). A recent revision of this genus has resulted in a phylogenetic hypothesis that distributes species among several genera (Hutchinson et al. 1990; Hutchinson and Donnellan, 1992) and thus differs from prior hypotheses (Greer, 1974, 1982). Pseudemoia entrecasteauxii has a structurally elaborate chorioallantoic placenta (Harrison and Weekes, 1925; Weekes, 1930) and Weekes (1935) predicted that the species was matrotrophic on the basis of the small size of oviductal eggs. Bassiana duperreyi is an oviparous species and a close relative of P. entrecasteauxii in each of the competing phylogenetic hypotheses (Greer, 1974, 1982; Hutchinson et al. 1990). Our primary goal was to test Weekes (1935) hypothesis that P. entrecasteauxii is matrotrophic. Additionally, we provide the first comparison of the sources of embryonic nutrition in two closely related reptile species with different modes of reproduction. This comparison offers support for the hypothesis (Weekes, 1935) that the evolution of placentotrophy in Squamata was associated with a concomitant reduction in egg size. Materials and methods Pseudemoia entrecasteauxii (Duméril and Bibron) were collected from Kanangra Boyd National Park, New South Wales. Recently ovulated eggs were removed from the right oviduct of 19 females collected in October, Neonates were obtained following

3 Embryonic nutrition in a viviparous reptile 99 parturition from a second series of 15 females collected from the same locality in January, Twelve female Bassiana duperreyi (Gray) were collected in southeastern Tasmania in November, 1990, returned to the University of Sydney and maintained until oviposition. The shell and contents of one egg from each clutch were separated and frozen on the day of oviposition. Embryos in the oviposited eggs contained lightly pigmented eyes, an open choroid fissure and paddle-like limbs; features corresponding to embryonic stages of Dufaure and Hubert (1961). Additional eggs were incubated at temperatures that fluctuated between 23.8 and 26.8 C on a mixture of 2:1 (water:vermiculite), which corresponds to a water potential greater than 100kPa, determined for that batch of Terra Lite Grade 3 vermiculite by thermocouple psychrometry using a Wescor HR-33T dewpoint microvoltmeter. Eighteen eggs, two in each of six clutches and one in each of the remaining six clutches, were incubated to hatching. Eggs and neonates of P. entrecasteauxii and shell-free egg contents and hatchlings of B. duperreyi were treated in the same manner for analyses. Specimens were lyophilized, and ashed in a muffle furnace at 550 C for 24h prior to hot acid digestion (Kopp and McKee, 1979) in preparation for inorganic ion analysis. Quantitative estimates of calcium, magnesium and phosphorus were carried out on an ARL inductively coupled plasma spectrophotometer (model 3510). Potassium and sodium levels were estimated using atomic absorption spectrophotometry (Perkin-Elmer model 2380). Differences between developmental stages within species were tested using either oneway analysis of variance (ANOVA) or two-way analysis of variance. Analysis of covariance (ANCOVA) was used for interspecific comparisons of fecundity and egg and offspring composition. Valus are presented as mean ± S.E. Results Mean snout vent length for the total sample of female P. entrecasteauxii was 58.8±0.8mm (range 48 68mm, N=34). Litter size, based on number of oviductal eggs and number of neonates (3.6±0.2, range 2 6, N=34) was positively correlated with female snout vent length [y= x; F(1,32)=8.6, P<0.01]. Neither egg nor neonate size was correlated with female size. Neonates contained significantly greater quantities of water, ash and organic molecules, estimated as dry mass minus ash, than did eggs (Table 1). Because of their small mass, oviductal eggs taken from individual females were pooled for inorganic analyses. Mean values per egg or neonate per litter were used to test for differences between egg and neonate composition for inorganic ions. Quantities of calcium, phosphorus, magnesium, sodium and potassium were significantly greater in neonates (Table 2). Mean snout vent length for female Bassiana duperreyi was 66.8±1.6mm (range mm, N=12). Clutch size (5.8±0.4, range 4 9) was correlated significantly with female snout vent length [y= x; F(1,10)=11.0, P<0.01]. Wet mass of shelled eggs sampled at oviposition (318.3±10.5mg) did not differ [F(1,22)=0.04] from the initial wet mass of shelled eggs incubated to hatching (315.5±9.8mg). The mean incubation time was 38±0.4days (range 34 41days). Wet mass and water content of hatchlings were

4 100 J. R. STEWART AND M. B. THOMPSON significantly greater than wet mass and water content of shell-free eggs (Table 3). However, hatchlings contained significantly less dry mass and organic mass than oviposited shell-free eggs. In contrast, significantly higher quantities of ash and calcium Table 1. Composition (mean ± S.E.) of oviductal eggs and neonates of Pseudemoia entrecasteauxii Eggs (N=19) (mg) Neonates (N=15) (mg) Wet mass 72.4±1.6* 286.5±3.2* Dry mass 32.5±0.8* 54.6±1.0* Water 39.9±1.0* 231.9±2.8* Organic mass 30.6±0.7* 49.3±0.9* Ash mass 1.9±0.08* 5.3±0.08* Asterisks indicate significant differences between columns; *P< See Appendix 1 for analysis of variance. Analysis of variance tables for Table 1. Appendix 1 Wet mass Source of variation SS d.f. MS Stage (egg/neonate) Female Litter (within female) Total Dry mass Source of variation SS d.f. MS Stage (egg/neonate) Female Litter (within female) Total Water mass Source of variation SS d.f. MS Stage (egg/neonate) Female Litter (within female) Total Organic mass Source of variation SS d.f. MS Stage (egg/neonate) Female Litter (within female) Total Inorganic mass Source of variation SS d.f. MS Stage (egg/neonate) Female Litter (within female) Total

5 Embryonic nutrition in a viviparous reptile 101 in hatchlings compared to eggs indicates that the shell is an important source of inorganic nutrients for developing embryos. These data compare favorably with a similar analysis for the North American oviparous scincid lizard Eumeces fasciatus (Florian, 1990) and support prior work demonstrating that the shell of oviparous squamate reptiles is an important source of embryonic calcium (Packard and Packard, 1984, 1988; Packard et al. 1985). Table 2. Inorganic composition (mean per egg/neonate per litter ± S.E.) of oviductal eggs and neonates of Pseudemoia entrecasteauxii Eggs (N=19) (mg) Neonates (N=15) (mg) Calcium 0.30±0.02** 1.09±0.02** Phosphorus 0.44±0.02** 0.75±0.02** Magnesium 0.03±0.001* 0.04±0.001* Sodium 0.09±0.009** 0.43±0.01** Potassium 0.07±0.008** 0.40±0.01** Asterisks indicate significant differences between columns; *P<0.005; **P< See Appendix 2 for analysis of variance. Analysis of variance tables for Table 2. Appendix 2 Calcium Source of variation SS d.f. MS Stage (egg/neonate) Residual Total Phosphorus Source of variation SS d.f. MS Stage (egg/neonate) Residual Total Magnesium Source of variation SS d.f. MS Stage (egg/neonate) Residual Total Sodium Source of variation SS d.f. MS Stage (egg/neonate) Residual Total Potassium Source of variation SS d.f. MS Stage (egg/neonate) Residual Total

6 102 J. R. STEWART AND M. B. THOMPSON Table 3. Composition (mean per egg/neonate per litter ± S.E.) and F values for the ANOVA for oviposited eggs and hatchlings of Bassiana duperreyi Source of variation Egg a Hatchling a (mg) (mg) Treatments b Blocks c Wet mass 245.5± ± * 2.1 NS Dry mass 79.8± ± *** 9.2*** Water 165.7± ± ** 1.5 NS Organic mass 76.4± ± *** 8.8*** Ash mass 3.4± ± *** 3.7* Calcium mass 0.44± ± *** 2.6 NS a N=12, except calcium mass, N=11. b Developmental stage. c Clutch. *P<0.05; **P<0.005; ***P<0.001; NS, not significant. See Appendix 3 for analysis of variance. Analysis of variance tables for Table 3. Appendix 3 Wet mass Source of variation SS d.f. MS Stage (egg/hatchling) Clutch Residual Total Dry mass Source of variation SS d.f. MS Stage (egg/hatchling) Clutch Residual Total Water Source of variation SS d.f. MS Stage (egg/hatchling) Clutch Residual Total Organic mass Source of variation SS d.f. MS Stage (egg/hatchling) Clutch Residual Total Ash mass Source of variation SS d.f. MS Stage (egg/hatchling) Clutch Residual Total

7 Embryonic nutrition in a viviparous reptile 103 Calcium mass Source of variation SS d.f. MS Stage (egg/hatchling) Clutch Residual Total Female B. duperreyi were significantly larger than female P. entrecasteauxii [F(1,44)=23.8; P<0.001]. Clutch size, adjusted for female size by ANCOVA, of B. duperreyi was significantly higher than litter size of P. entrecasteauxii (Table 4). Shell-free eggs of B. duperreyi contained greater quantities of all variables measured compared to the yolk of recently ovulated eggs of P. entrecasteauxii (Table 4). Dry mass and organic mass of hatchling B. duperreyi were significantly greater than those of neonatal P. entrecasteauxii. However, neonatal P. entrecasteauxii contained greater quantities of ash and water and had significantly greater wet mass (Table 4). Discussion Embryonic nutrition in P. entrecasteauxii In her comprehensive review of reptilian placentation, Weekes (1935) speculated that Pseudemoia entrecasteauxii (as Lygosoma entrecasteauxii) was matrotrophic. This hypothesis was based on the small size of oviductal eggs and the presence of a specialized chorioallantoic placenta in this species. Our estimates of net placental uptake confirm that P. entrecasteauxii is matrotrophic. Neonatal dry mass, as well as estimates of both organic and inorganic components of dry mass, exceeded that of recently ovulated eggs (Tables 1, 2). Further, although the pattern of embryonic metabolism and thus the extent of placental transport of organic molecules, is unknown, the degree of matrotrophy in this species is Table 4. Mean composition (mg) adjusted for female snout vent length and F values for the ANCOVA for eggs and offspring of Bassiana duperreyi and Pseudemoia entrecasteauxii B. duperreyi P. entrecasteauxii F value Clutch/litter size * Egg wet mass *** Egg dry mass *** Egg water *** Egg organic mass *** Egg ash *** Offspring wet mass * Offspring dry mass *** Offspring water ** Offspring organic mass *** Offspring ash ** *P<0.05; **P<0.005; ***P<0.001.

8 104 J. R. STEWART AND M. B. THOMPSON substantial in comparison to that of many viviparous squamates (Stewart, 1992). Most viviparous species give birth to neonates with less dry mass than the large yolked eggs they ovulate (Thompson, 1977, 1981, 1982; Stewart and Castillo, 1984; Stewart, 1989; Stewart et al. 1990), although each of these species relies on some degree of placentotrophy, at least for inorganic molecules. The degree of matrotrophy in P. entrecasteauxii is impressive, yet embryonic gains in dry mass are much less than those recorded for two other scincid lizards. Dry mass of neonates of Mabuya heathii and M. bistriata is % greater than egg dry mass (Blackburn et al. 1984; Vitt and Blackburn, 1991). A comparison of egg and offspring dry mass among scincid lizards (Table 5) indicates that P. entrecasteauxii is intermediate in embryonic nutritional pattern in comparison to Eulamprus quoyii and the two species of Mabuya. For example, E. quoyii gives birth to neonates with less dry mass than its ovulated eggs, yet embryos do incorporate inorganic ions from placental sources in excess of yolk reserves (Thompson, 1977, 1982). The pattern of net placental transfer of ions for E. quoyii is similar to that of P. entrecasteauxii. However, the relative quantities provided by the placenta are greater in P. entrecasteauxii. Thus, 56% of calcium incorporated into E. quoyii neonates is provided from the placentae (Thompson, 1977), whereas placentotrophy in P. entrecasteauxii accounts for 72% of neonatal calcium (Table 2). The pattern of embryonic nourishment in P. entrecasteauxii differs from that of E. quoyii by placing less reliance on lecithotrophy and greater reliance on placentotrophy. The mean dry mass of hatchling B. duperreyi was 85% and organic mass was 83% of shell-free egg values (Table 3). If the conversion ratio for P. entrecasteauxii development is comparable, i.e. the effects of scale due to egg size and source of energy are minimal, then yolk provides 51% (30.6mg; Table 1) of the organic material for growth and metabolism, with the remaining 49% (29.0mg) being transferred across the placenta. This estimate assumes that it takes 59.6mg of organic material to produce a neonate with an organic mass of 49.3mg (Table 1). In contrast, nearly all embryonic nutrients are transferred across the uterus in M. heathii and M. bistriata. Information on embryonic nutrition among viviparous squamates is scant, particularly in view of the great number of viviparous species. Nonetheless, the pattern that is emerging is that placentotrophy occurs in all viviparous squamate species, regardless of Table 5. Egg dry mass and offspring dry mass for lizards of the family Scincidae Egg Offspring Offspring/ (mg) (mg) egg Eumeces fasciatus a (O) Bassiana duperreyi (O) Eulamprus quoyii b (V) Pseudemoia entrecasteauxii (V) Mabuya heathi c (V) Mabuya bistriata d (V) a Florian (1990); b Thompson (1977); c Blackburn et al. (1984); d Vitt and Blackburn (1991). O, oviparous; V, viviparous.

9 Embryonic nutrition in a viviparous reptile 105 the quantity of yolk present at ovulation (Blackburn, 1992; Stewart, 1992). The degree of placentotrophy in P. entrecasteauxii is unique among squamate species that have been studied in that yolk and placental nourishment are approximately equivalent. Comparative embryonic nutrition in B. duperreyi and P. entrecasteauxii Two hypotheses have been proposed for phylogenetic relationships among Australian lizard species assigned to the scincid genus Leiolopisma (Greer, 1974, 1982; Hutchinson et al. 1990). Greer (1974) retained the generic designation Leiolopisma and subdivided the included species into two groups. The Baudini group included L. entrecasteauxii and L. duperreyi. Hutchinson et al. (1990) assigned L. entrecasteauxii to the genus Pseudemoia and L. duperreyi to the genus Bassiana, yet hypothesized a common ancestor for these two taxa. Although there are substantive differences between these hypotheses, they are consistent in arguing for a close phylogenetic relationship between P. entrecasteauxii and B. duperreyi. Variation in lizard life history patterns results from covariation among specific life history characteristics (Tinkle et al. 1970; Dunham et al. 1988). In general, viviparous species are late maturing, long lived and produce a single litter per reproductive season. Fecundity is higher in oviparous species that produce multiple clutches per season (Tinkle et al. 1970) and clutch size is larger in single-brooded oviparous species than in viviparous species (Dunham et al. 1988). Compared to B. duperreyi, P. entrecasteauxii ovulates fewer, smaller eggs (Table 4). An average litter, adjusted for female size, for P. entrecasteauxii contains one less egg that has only 42% of the dry mass of an average B. duperreyi egg. Reduced clutch size in a viviparous species of lizard compared to a closely related oviparous species has also been demonstrated within the family Iguanidae (Guillette, 1982). Within these two lineages, the evolution of viviparity is associated with a reduction in clutch size. Greater maternal commitment to individual young has been viewed as a characteristic of viviparity in comparison to oviparity (Shine, 1978; Guillette, 1982; Stewart, 1989). In support of this, both egg and neonate size were greater in Sceloporus bicanthalis, a viviparous species, compared to the oviparous S. aeneus (Guillette, 1981, 1982). In contrast, both eggs and hatchlings of B. duperreyi had greater dry mass, relative to female size, than eggs and neonates of P. entrecasteauxii (Table 4). However, two estimates of offspring content did indicate greater provision to individual embryos in P. entrecasteauxii as a result of uterine gestation. The total inorganic mass of P. entrecasteauxii neonates exceeded that of hatchling B. duperreyi. Additionally, embryos of the viviparous species were more efficient in the uptake, or at least the retention, of water, in spite of the substratum water potentials during incubation of B. duperreyi eggs being at levels considered high for squamate egg incubation (Packard et al. 1982; Packard and Packard, 1987). Using calculations based on B. duperreyi, in the absence of placental nourishment, P. entrecasteauxii neonates would contain 25.3mg of organic mass and 27.7mg of dry mass. These neonates would be roughly half the size of those born into the population we sampled. Experimental reduction in yolk quantity, by up to 50%, in eggs of an oviparous iguanid lizard resulted in smaller, yet viable, hatchlings (Sinervo, 1990). Thus,

10 106 J. R. STEWART AND M. B. THOMPSON matrotrophy in P. entrecasteauxii may not be a physiological requirement for the production of viable neonates. An equally significant possibility is that lecithotrophy may not be necessary for the production of viable neonates. However, in the absence of matrotrophy, the production of smaller numbers of markedly smaller young would result in significant life history consequences. Supplementation of yolk nourishment by placentotrophy is characteristic of viviparous squamate species that ovulate much larger ova than P. entrecasteauxii (Stewart, 1992). Thus, P. entrecasteauxii resembles other species in supplementing yolk nourishment by placentotrophy; it is the much greater degree of placentotrophy that is notable. Although the eggs of P. entrecasteauxii were much smaller than those of B. duperreyi, the size disparity at parition was greatly reduced as a result of uterine gestation (Table 4). There are two possible historical sequences that could have resulted in the reproductive pattern of P. entrecasteauxii. Weekes (1935) hypothesis was that egg content had undergone evolutionary reduction in quantity in association with an increased reliance on placental nourishment and, by implication, neonate size remained constant. Alternatively, egg content in the ancestors of P. entrecasteauxii did not differ from that in present populations, but neonates were much smaller. The advent of placentotrophy thus produced an evolutionary increase in neonatal size. We propose that the most likely historical sequence is that offspring size has been conserved, that is, the ancestors of P. entrecasteauxii ovulated larger eggs, comparable to those of B. duperreyi and that the evolution of matrotrophy has been accompanied by a concomitant reduction in yolk quantity. The alternative hypothesis requires that the ancestor of both species had small eggs and that both lineages, representing two reproductive modes, converged on offspring size through two separate patterns of embryonic nutrition. That is, yolk content increased in the B. duperreyi lineage and a functional placenta evolved in P. entrecasteauxii. The mode of embryonic nourishment in P. entrecasteauxii is unique among squamates in its balance between lecithotrophy and placentotrophy and contributes significantly to our understanding of variation in reptilian reproductive biology. The relationship between reproductive mode and embryonic nutrition is now known to occur in four patterns among Reptilia; (1) oviparous and lecithotrophic, (2) viviparous and predominantly lecithotrophic, (3) viviparous and equivalent contributions from lecithotrophy and placentotrophy and (4) viviparous and predominantly placentotrophic. It is also important that the mode of embryonic nutrition shown by P. entrecasteauxii supports a gradualistic model for the evolution of placentotrophy among reptiles. The roughly equal contribution from ovarian and uterine sources is functionally intermediate between lecithotrophy and matrotrophy regardless of the yolk content of ancestral eggs. This project was supported, in part, by the Office of Research, University of Tulsa, and The School of Biological Sciences, University of Sydney. Approval for the project was provided by The University of Sydney Animal Care and Ethics Committee, approval no. 90/L04/20. Animals were collected under permit no. B783 from the New South Wales National Parks and Wildlife Service. Specimens were deposited in the Australian Museum. We are grateful to the many people who contributed in the field and laboratory

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